Although evidence suggests that mitochondrial dysfunction stimulates the production of reactive oxygen species (ROS) that trigger dopaminergic cell death in Parkinson's disease (PD) the molecular mechanisms responsible for mitochondrial ROS production are unknown. We have recently discovered that the multi-subunit enzyme alpha-ketoglutarate dehydrogenase (alpha-KGDC) is a substantial source of ROS production in brain mitochondria. The activity and immunoreactivity of this protein has been shown to be altered in neurons and in the brains of animals treated with MPP' and in the substantia nigra of patients with PD. We hypothesize that dysregulation in the intramolecular electron transfer within the subunits of alpha-KGDC is a primary mediator of oxidative stress associated with PD and to ROS-mediated neuronal cell death. The specific aims of this exploratory project are: 1. Quantify the contribution of alpha-KGDC to ROS production in isolated brain mitochondria in the absence and presence of PD-associated neurotoxins. We will compare the ROS production by alpha-KGDC, other mitochondrial dehydrogenases and electron transport chain Complex I. We will also determine if the ROS generated by alpha-KGDC and Complex I interact to decrease normal enzyme activity while increasing production of ROS. 2. Explore possible chemical mechanisms of ROS production by different enzyme subunits of the alpha-KGDC. 3. Develop a cell culture model for assessing the contribution of alpha-KGDC to oxidative stress and the interactions of alpha-KGDC and Complex I in the absence and presence of neurotoxins. We will measure the effects of MPP' in the absence and presence of high extracellular alpha-ketoglutarate and alpha-KGDC inhibitors on markers of protein and DNA oxidation. The effects of different culture conditions on alpha-KGDC and Complex I enzyme activities and on H2O2 production will be measured using mitochondria isolated from these cells. This project will lay the foundation for the molecular etiology of cell death in PD which could be activated by genetic and (or) environmental determinants. Verification of the role of alpha-KGDC in mitochondrial ROS generation and in oxidative cell death could lead to the development of genetic animal models of susceptibility to PD disease leading to the development of targeted neuroprotective interventions that will minimize the incidence or slow the progression of Parkinson's disease.